S100B mini-promoters

ABSTRACT

Isolated polynucleotides comprising an S100B promoter are provided, where an S100B regulatory element is operably joined to an S100B basal promoter utilizing a non-native spacing between the promoter and regulatory elements. The promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, etc.

FIELD OF THE INVENTION

The invention relates to gene promoters and regulatory elements. Morespecifically, the invention relates to novel S100B promoter compositionsand related methods.

BACKGROUND

S100B is a member of the S100 protein family. S100 protein is a lowmolecular weight protein found in vertebrates characterized by twocalcium binding sites of the helix-loop-helix (“EF-hand type”)conformation. There are at least 21 different types of S100 proteins.The name is derived from the fact that the protein is 100% soluble inammonium sulfate at neutral pH. S100B is an acidic protein with amolecular weight of 21 kDa existing as a homodimer consisting of twobeta subunits. The two monomers are configured in a twofold axis ofrotation and are held together by disulfide bonds. S100B is involved inthe regulation of energy metabolism in brain cells. S100B is producedprimarily by astrocytes and exerts autocrine and paracrine effects onglia, neurons, and microglia. Furthermore, it interacts with manyimmunological functions of the brain (reviewed in Rothermundt et al.2003).

Functional S100B promoter sequences have been identified and analyzed inseveral mammalian species. The structure of the human S100B gene andproximal promoter region was initially elucidated by Allore (1990).Castets et al. (1997) analyzed several human constructs containingdifferent fragments of the human S100B promoter which revealed a complexpattern of regulation relating to different regions of the promoter. Inthis study, a number of positive and negative regulatory elements thatare at least partially involved in regulating cell specific expressionwere identified in the region between −1012 and +697 (with +1 beingdefined as the first nucleotide in exon 1 of the S100B gene).Additionally, a negative regulatory element is thought to be located inthe region between −4437 and −1012 of the upstream region, however theprecise location of this element was not elucidated. This studyhighlighted the complexity of the regulation of this gene to allowspecific cell specific expression of human S100B. Lin et al. (2004)analyzed p53 transcription factor binding sites and their effect onhuman S100B promoter activity in malignant melanoma cells.

Expression of S100B has been shown to be spatiotemporally associatedwith maturation of glial cells in mice (Deloulme et al. 2004; Raponi etal. 2007) and rats (Hagiwara and Sueoka 1995). In a separate study, anenhanced green fluorescent protein (EGFP) reporter was fused to the−1669/+3116 region of the mouse S100B gene, resulting in observedexpression in both astrocytes and oligodendrocytes, an expressionpattern that occurred in both a spatial and temporal fashion duringmouse brain development (Vives et al. 2003). More recent data using thissame expression construct further highlights the spatiotemporalexpression pattern in both astrocytic and oligodendrocytic lineages inthe mouse brain (Hachem et al. 2005). Analysis of expression patterns inhumans have also revealed spatiotemporal expression during braindevelopment, particularly in proliferating and mature astrocytes(Marshak 1990; Tiu et al. 2000). Antibodies raised to S100B have beenshown to label primarily astrocytes in human brain (Lyck et al. 2008).

There exists a significant need for promoter elements which are capableof driving expression in specific cell types and/or in specific regionsof the brain. Identification of minimal elements required for adequateexpression and specificity will allow ease of use in expressionconstructs.

SUMMARY OF THE INVENTION

The present invention provides novel nucleic acid sequence compositionsand methods, which relate to S100B promoters having a sequence otherthan a native S100B promoter.

In one embodiment of the invention, there is provided an isolatednucleic acid fragment comprising an S100B mini-promoter, wherein theS100B mini-promoter comprises an S100B regulatory element operablylinked in a non-native conformation to an S100B basal promoter. TheS100B promoter may have a nucleic acid sequence that is substantiallysimilar in sequence and function to SEQ ID NO: 1. The S100B regulatoryelement may have a nucleic acid sequence that is substantially similarin sequence and function to SEQ ID NO: 2. The S100B basal promoter mayhave a nucleic acid sequence that is substantially similar in sequenceand function to SEQ ID NO: 3. The S100B promoter may further be operablylinked to an expressible sequence, e.g. reporter genes, genes encoding apolypeptide of interest, regulatory RNA sequences such as miRNA, siRNA,anti-sense RNA, etc., and the like. Reporter gene sequences include, forexample luciferase, beta-galactosidase, green fluorescent protein,enhanced green fluorescent protein, and the like as known in the art.The expressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expressible sequence may encode an RNA interference molecule.

In one embodiment, there is provided an expression vector comprising anS100B mini-promoter element, wherein the S100B mini-promoter elementcomprises an S100B regulatory element operably linked in a non-nativeconformation to an S100B basal promoter element. The S100B promoter mayhave a nucleic acid sequence which is substantially similar in sequenceand function to SEQ ID NO: 1. The S100B regulatory element may have anucleic acid sequence which is substantially similar in sequence andfunction to SEQ ID NO: 2. The S100B basal promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 3. The S100B promoter may further be operably linked to anexpressible sequence, e.g. reporter genes, genes encoding a polypeptideof interest, regulatory RNA sequences such as miRNA, siRNA, anti-senseRNA, etc., and the like. Reporter gene sequences include, for exampleluciferase, beta-galactosidase, green fluorescent protein, enhancedgreen fluorescent protein, and the like as known in the art. Theexpressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, and the like.The expressible sequence may encode an RNA interference molecule. Theexpression vector may further comprise a genomic targeting sequence. Thegenomic targeting sequence may be HPRT.

In one embodiment, there is provided a method for selective expressionof a gene, protein, RNA interference molecule or the like in a cell, themethod comprising introducing into the cell a expression vectorcomprising an S100B mini-promoter element of the invention, wherein theS100B mini-promoter element comprises an S100B regulatory elementoperably linked in a non-native conformation to an S100B basal promoterelement. Cells of interest include, without limitation, cells of theperipheral or central nervous system and progenitors thereof, e.g.embryonic stem cells, neural stem cells, neurons, glial cells,astrocytes, microgial cells, etc. The S100B promoter may have a nucleicacid sequence which is substantially similar in sequence and function toSEQ ID NO: 1. The S100B regulatory element may have a nucleic acidsequence which is substantially similar in sequence and function to SEQID NO: 2. The S100B basal promoter may have a nucleic acid sequencewhich is substantially similar in sequence and function to SEQ ID NO: 3.The S100B promoter may further be operably linked to an expressiblesequence, e.g. reporter genes, genes encoding a polypeptide of interest,regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., andthe like. Reporter gene sequences include, for example luciferase,beta-galactosidase, green fluorescent protein, enhanced greenfluorescent protein, and the like as known in the art. The expressiblesequence may encode a protein of interest, for example a therapeuticprotein, receptor, antibody, growth factor, and the like. Theexpressible sequence may encode an RNA interference molecule. Theexpression vector may thus further comprise a genomic targetingsequence. The genomic targeting sequence may be HPRT.

In one embodiment of the invention, there is provided a method foridentifying or selectively labeling a cell, the method comprisingintroducing into the cell a expression vector comprising an S100Bmini-promoter element operably linked to an expressible sequence,wherein the S100B mini-promoter element comprises an S100B regulatoryelement operably linked in a non-native conformation to an S100B basalpromoter element, and wherein the expressible sequence comprises areporter gene. The S100B promoter element may have a nucleic acidsequence substantially similar in sequence and function to SEQ ID NO: 1.The S100B regulatory element may have a nucleic acid sequencesubstantially similar in sequence and function to SEQ ID NO: 2. TheS100B basal promoter element may have a nucleic acid sequencesubstantially similar in sequence and function to SEQ ID NO:3. In someembodiments, the cell is a peripheral or central nervous system cell orprogenitors thereof, including, without limitation, embryonic stemcells, neural stem cells, glial cell, e.g. Bergmann glial cells,astrocytes, and the like. Reporter gene sequences include, for exampleluciferase, beta-galactosidase, green fluorescent protein, enhancedgreen fluorescent protein, and the like as known in the art. Theexpressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, RNA interferencemolecule and the like.

In one embodiment of the invention, there is provided a method formonitoring or tracking the development or maturation of a cell, themethod comprising: 1) introducing into the cell a expression vectorcomprising an S100B mini-promoter element operably linked to anexpressible sequence, wherein the S100B mini-promoter element comprisesan S100B regulatory element operably linked in a non-native conformationto an S100B basal promoter element, and wherein the expressible sequencecomprises a reporter gene; and 2) detecting the expression of thereporter gene in the cell of in progeny of the cell as a means ofdetermining the lineage, identity or developmental state of the cell orcell progeny. The S100B promoter element may have a nucleic acidsequence substantially similar in sequence and function to SEQ ID NO: 1.The S100B regulatory element may have a nucleic acid sequencesubstantially similar in sequence and function to SEQ ID NO: 2. TheS100B basal promoter element may have a nucleic acid sequencesubstantially similar in sequence and function to SEQ ID NO: 3. In someembodiments, the cell into which the expression vector is initiallyintroduced is a peripheral or central nervous system cell or progenitorsthereof, including, without limitation, embryonic stem cells, neuralstem cells, glial cell, e.g. Bergmann glial cells, astrocytes, and thelike.

SHORT DESCRIPTION OF FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. From top to bottom, the human genomic sequence of S100B locatedon chromosome 21 with an arrow pointing to the transcription start site,the gene exons as black boxes, the non-coding conserved regions as blueboxes with open black boxes defining the promoter elements referred toin the present invention, the conservation profile between the human andmouse S100B sequences with the grey area delineating the 54% thresholdused.

FIG. 2—DNA expression vector (pEMS1306) into which S100B promoterelements were inserted for expression studies. The S100B promoter with anucleic acid sequence corresponding to SEQ ID NO: 1 was inserted intothe multiple cloning site (MCS) of the pEMS1306 vector such that itbecame operably linked to the enhanced green fluorescent protein (EGFP)reporter gene. The final construct, called S100B-C, also contained theHPRT genomic targeting sequence, an ampicillin resistance gene (AmpR)for screening, and a transcriptional termination sequence (SV40 polyA),as well as other elements necessary for vector replication and geneexpression.

FIG. 3. A 14.5% chimera (tEMS 1561) from the S100B-C minipromoter(pEMS1384) strain shows specific staining in the cerebellum, labelingBergman glial cells. A) Brightfield micrograph from the cerebellum.Anti-GFP staining (brown DAB reaction product) reveals radial processesof Bergman glia as well as in the cell bodies (small arrowheads) andpial endfeet (large arrowheads) in a clearly chimeric pattern (i.e. onlya subpopulation of cells are labeled). B) Confocal laser scanningmicrograph of a thin optical section in the cerebellum. Double label inthe cerebellum of GFP fluorescence (green) and anti-GFAPimmunofluorescence (red) clearly shows similar chimeric staining in theBergman glia. GFP fluorescence highlights Bergman glial cell bodies(small arrowheads) and decorates processes (large arrowheads) thatfollow the GFAP (red) positive GFAP-positive radial processes. PCL,Purkinje cell layer; ML Molecular Layer, pia, apical pial surface.

DETAILED DESCRIPTION

The polynucleotide compositions of the present invention comprise anovel arrangement of S100B promoter elements (also referred to herein asS100B mini-promoters) as well as novel expression vectors comprisingsaid arrangement of S100B promoter elements (or mini-promoters). Thepresent invention also includes various methods of utilizing these novelS100B promoter (or mini-promoter) elements or expression vectors.

Provided is a sequence listing including certain of the S100Bmini-promoters, wherein SEQ ID NO:1 comprises the human S100Bmini-promoter (3892 bp). Nucleotides 1-2507 comprise the human S100Bregulatory element; and nucleotides 2508-3892 comprise the human S100Bbasal promoter element. SEQ ID NO: 2 comprises the human S100Bregulatory element, which corresponds to human genome position: chr.2146867265-46864758. SEQ ID NO:3 comprises the human S100B basal promoterelement, which corresponds to human genome position: chr.2146850766-46849383.

The term ‘S100B’ refers to the gene that encodes the S100B protein, andincludes the controlling regulatory elements, e.g. promoters and thelike. S100B is referred to as S100beta, S100 calcium binding proteinbeta, S100 calcium binding protein B, and NEF. The protein encoded bythis S100B is a member of the S100 family of proteins containing 2EF-hand calcium-binding motifs. S100 proteins are localized in thecytoplasm and/or nucleus of a wide range of cells, and involved in theregulation of a number of cellular processes such as cell cycleprogression and differentiation. The human homolog of S100B is encodedby the human gene identified as EntrezGene #6285, and is located atchromosomal location 21q22.3. The protein encoded by human S100B has theProtein Accession #P04271 (Swiss-Prot). Other mammalian S100B homologsinclude but are not limited to: Rattus norvegicus (EntrezGene #25742,Protein Accession #P04631), Mus musculus (EntrezGene #20203, ProteinAccession #P50114).

The term ‘promoter’ refers to the regulatory DNA region which controlstranscription or expression of a gene and which can be located adjacentto or overlapping a nucleotide or region of nucleotides at which RNAtranscription is initiated. A promoter contains specific DNA sequenceswhich bind protein factors, often referred to as transcription factors,which facilitate binding of RNA polymerase to the DNA leading to genetranscription. A ‘basal promoter’, also referred to as a ‘corepromoter’, generally refers to a promoter that contains all the basicnecessary elements to promote transcriptional expression of an operablylinked polynucleotide. Eukaryotic basal promoters typically, though notnecessarily, contain a TATA-box and/or a CAAT box. An ‘S100B basalpromoter’, in the context of the present invention and as used herein,is a nucleic acid compound having a sequence with at least 65%, at least70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least99% similarity to SEQ ID NO: 3, and which comprises at least 5, usuallyat least 8, and may comprise all 11 of the identified conservedsequences listed in Table 1. The S100B basal promoters of the presentinvention may comprise a TATA box such as that found at position −29(relative to the transcriptional start site) of the native human S100Bpromoter and/or a CAAT box (in this case, a reverse CAAT box) such asthat found at position −93 (relative to the transcriptional start site)of the native human S100B promoter, and these elements should bepositioned relative to the transcriptional start site (+1) in a way thatis reflective of the native sequence.

TABLE 1 List of conserved sequences in the human S100B basal promoter -SEQ ID NO: 3. Start (relative to End (relative to SEQ ID NO: 3) SEQ IDNO: 3) Invariant sequence type 79 116 p53 validated binding site 246 285Conserved sequence 424 503 Conserved sequence 562 585 Conserved sequence769 792 Conserved sequence 825 844 Conserved sequence 880 933 Conservedsequence 949 968 Conserved sequence 975 1146 Conserved sequence 11541173 Conserved sequence 1192 1218 Conserved sequence The start and endcoordinates of the sequences are relative to the full SEQ ID NO: 3sequence.

A promoter may also include ‘regulatory elements’ which may alsoinfluence the expression or transcription by the promoter. Suchregulatory elements encode specific DNA sequences which bind otherfactors, which may include but are not limited to enhancers, silencers,insulators, and/or boundary elements. An ‘S100B regulatory element’, inthe context of the present invention and as used herein, is a nucleicacid compound having a sequence with at least 65%, at least 70%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%similarity to SEQ ID NO: 2, and which comprises at least 20, usually atleast 25, and may comprise all 27 of the identified conserved sequenceslisted in Table 2. The present invention provides, in certainembodiments as described herein, different promoters of the S100B gene.In some embodiments, the S100B promoter comprises an S100B regulatoryelement operably linked to an S100B basal promoter.

TABLE 2 List of conserved sequences in the human S100B regulatoryelement - SEQ ID NO: 2. Start (relative to End (relative to SEQ ID NO:2) SEQ ID NO: 2) Invariant sequence type 85 105 Conserved sequence 141160 Conserved sequence 212 231 Conserved sequence 317 344 Conservedsequence 415 447 Conserved sequence 495 514 Conserved sequence 531 553Conserved sequence 562 582 Conserved sequence 603 624 Conserved sequence651 710 Conserved sequence 720 746 Conserved sequence 806 829 Conservedsequence 931 966 Conserved sequence 1191 1210 Conserved sequence 12451287 Conserved sequence 1300 1341 Conserved sequence 1350 1375 Conservedsequence 1422 1445 Conserved sequence 1548 1577 Conserved sequence 16301658 Conserved sequence 1708 1730 Conserved sequence 1743 1783 Conservedsequence 1786 1807 Conserved sequence 1912 1938 Conserved sequence 20882158 Conserved sequence 2212 2231 Conserved sequence 2315 2341 Conservedsequence The start and end coordinates of the sequences are relative tothe full SEQ ID NO: 2 sequence.

The term ‘operably linked’, in the context of the present invention,means joined in such a fashion as to work together to allowtranscription. In some embodiments of the invention, two polynucleotidesequences may be operably linked by being directly linked via anucleotide bond. In this fashion, the two operably linked elementscontain no intervening sequences and in being joined are able to directtranscription of an expression sequence. In other embodiments of theinvention, two elements may be operably linked by an interveningcompound, for instance a polynucleotide sequence of variable length. Insuch a fashion, the operably linked elements, although not directlyjuxtaposed, are still able to direct transcription of an expressionsequence. Thus, according to some embodiments of the invention, one ormore promoter elements may be operably linked to each other, andadditionally be operably linked to a downstream expression sequence,such that the linked promoter elements are able to direct expression ofthe downstream expression sequence.

The term ‘mini-promoter’ refers to a promoter in which certain promoterelements are combined in a non-native conformation, usually in such afashion as to reduce the overall size of the promoter compared to thenative conformation. For example, after identification of criticalpromoter elements, using one or more of various techniques, the nativesequences that intervene the identified elements may be partially orcompletely removed. Other non-native sequences may optionally beinserted between the identified promoter elements. A mini-promoter mayprovide certain advantages over native promoter conformations. Forexample, the smaller size of the mini-promoter may allow easier geneticmanipulation, ie. for the design and/or construction of expressionvectors or other recombinant DNA constructs. In addition, the smallersize may allow easier insertion of DNA constructs into host cells and/orgenomes, ie. via transfection, transformation, etc. Other advantages ofmini-promoters would be apparent to one of skill in the art. In someembodiments of the invention, there are thus provided novel S100Bmini-promoters comprising an S100B regulatory element operably linked ina non-native conformation to an S100B basal promoter. In general thespacing between the S100B regulatory element and the S100B basalpromoter is not more than about 15 KB, generally not more than about 10KB, usually not more than about 1 KB, more often not more than about 500nt, and may be not more than about 100 nt, and includes a direct joiningof the two sequences.

The term ‘expressible sequence’ refers to a polynucleotide that isoperably linked to a promoter element, such that the promoter elementcauses transcriptional expression of the expression sequence. Anexpressible sequence is typically linked downstream, on the 3′-end ofthe promoter element(s) in order to achieve transcriptional expression.The result of this transcriptional expression is the production of anRNA macromolecule. The expressed RNA molecule may encode a protein andmay thus be subsequently translated by the appropriate cellularmachinery to produce a polypeptide protein molecule. In some embodimentsof the invention, the expression sequence may encode a reporter protein.Alternately, the RNA molecule may be an antisense, RNAi or othernon-coding RNA molecule, which may be capable of modulating theexpression of specific genes in a cell, as is known in the art.

The term ‘RNA’ as used in the present invention includes full-length RNAmolecules, which may be coding or non-coding sequences, fragments, andderivatives thereof. For example, a full-length RNA may initiallyencompass up to about 20 Kb or more of sequence, and frequently will beprocessed by splicing to generate a small mature RNA. Fragments, RNAi,miRNA and anti-sense molecules may be smaller, usually at least about 18nt. in length, at least about 20 nt in length, at least about 25 nt. inlength, and may be up to about 50 nt. in length, up to about 100 nt inlength, or more. RNA may be single stranded, double stranded, synthetic,isolated, partially isolated, essentially pure or recombinant. RNAcompounds may be naturally occurring, or they may be altered such thatthey differ from naturally occurring RNA compounds. Alterations mayinclude addition, deletion, substitution or modification of existingnucleotides. Such nucleotides may be either naturally occurring, ornon-naturally occurring nucleotides. Alterations may also involveaddition or insertion of non-nucleotide material, for instance at theend or ends of an existing RNA compound, or at a site that is internalto the RNA (ie. between two or more nucleotides).

The term ‘nucleic acid’ as used herein includes any nucleic acid, andmay be a deoxyribonucleotide or ribonucleotide polymer in either singleor double-stranded form. A ‘polynucleotide’ or ‘nucleotide polymer’ asused herein may include synthetic or mixed polymers of nucleic acids,both sense and antisense strands, and may be chemically or biochemicallymodified or may contain non-natural or derivatized nucleotide bases, aswill be readily appreciated by those skilled in the art. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.),charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.),pendent moieties (e.g., polypeptides), and modified linkages (e.g.,alpha anomeric polynucleotides, etc.). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions.

A ‘purine’ is a heterocyclic organic compound containing fusedpyrimidine and imidazole rings, and acts as the parent compound forpurine bases, adenine (A) and guanine (G). ‘Nucleotides’ are generally apurine (R) or pyrimidine (Y) base covalently linked to a pentose,usually ribose or deoxyribose, where the sugar carries one or morephosphate groups. Nucleic acids are generally a polymer of nucleotidesjoined by 3′ 5′ phosphodiester linkages. As used herein ‘purine’ is usedto refer to the purine bases, A and G, and more broadly to include thenucleotide monomers, deoxyadenosine-5′-phosphate anddeoxyguanosine-5′-phosphate, as components of a polynucleotide chain. A‘pyrimidine’ is a single-ringed, organic base that forms nucleotidebases, such as cytosine (C), thymine (T) and uracil (U). As used herein‘pyrimidine’ is used to refer to the pyrimidine bases, C, T and U, andmore broadly to include the pyrimidine nucleotide monomers that alongwith purine nucleotides are the components of a polynucleotide chain.

It is within the capability of one of skill in the art to modify thesequence of a promoter nucleic acid, e.g. the provided basal promoterand regulatory sequences, in a manner that does not substantially changethe activity of the promoter element, i.e. the transcription rate of anexpressible sequence operably linked to a modified promoter sequence isat least about 65% the transcription rate of the original promoter, atleast about 75% the transcription rate of the original promotersequence, at least about 80%, at least about 90%, at least about 95%, atleast about 99%, or more in a selected cell or suitable in vitroenvironment. Such modified sequences would be considered to be‘functionally similar’ or to have ‘functional similarity’ or‘substantial functional similarity’ to the unmodified sequence. Suchmodifications may include insertions, deletions which may be truncationof the sequence or internal deletions, or substitutions. The level ofsequence modification to an original sequence will determine the‘sequence similarity’ of the original and modified sequences.Modification of the promoter elements of the present invention in afashion that does not significantly alter transcriptional activity, asdescribed above would result in sequences with ‘substantial sequencesimilarity’ to the original sequence i.e. the modified sequence has anucleic acid composition that is at least about 65% similar to theoriginal promoter sequence, at least about 75% similar to the originalpromoter sequence, at least about 80%, at least about 90%, at leastabout 95%, at least about 99%, or more similar to the original promotersequence. Thus, mini-promoter elements which have substantial functionaland/or sequence similarity are herein described and are within the scopeof the invention.

The nucleic acid sequence compositions provided in some embodiments ofthe invention contain p53 transcription factor binding sites, and itwould be reasonable to expect that the binding sequence at these sitesshould not be altered if one expects to maintain the similartranscriptional expression from the unmodified sequence. For instance,the S100B regulatory element of the invention corresponding to SEQ IDNO: 3 contains a p53 transcription factor binding site at position79-116 of SEQ ID NO: 3.

An ‘RNA interference molecule’, or ‘RNA interference sequence’ asdefined herein, may include, but is not limited to, an antisense RNAmolecule, a microRNA molecule or a short hairpin RNA (shRNA) molecule.Typically, RNA interference molecules are capable of target-specificmodulation of gene expression and exert their effect either by mediatingdegradation of the mRNA products of the target gene, or by preventingprotein translation from the mRNA of the target gene. The overall effectof interference with mRNA function is modulation of expression of theproduct of a target gene. This modulation can be measured in ways whichare routine in the art, for example by Northern blot assay or reversetranscriptase PCR of mRNA expression, Western blot or ELISA assay ofprotein expression, immunoprecipitation assay of protein expression,etc.

An ‘antisense RNA molecule’, as used herein, is typically a singlestranded RNA compound which binds to complementary RNA compounds, suchas target mRNA molecules, and blocks translation from the complementaryRNA compounds by sterically interfering with the normal translationalmachinery. Specific targeting of antisense RNA compounds to inhibit theexpression of a desired gene may design the antisense RNA compound tohave a homologous, complementary sequence to the desired gene. Perfecthomology is not necessary for inhibition of expression. Design of genespecific antisense RNA compounds, including nucleotide sequenceselection and additionally appropriate alterations, are known to one ofskill in the art.

The term ‘microRNA molecule’, ‘microRNA’ or ‘miRNA’, as used herein,refers to single-stranded RNA molecules, typically of about 21-23nucleotides in length, which are capable of modulating gene expression.Mature miRNA molecules are partially complementary to one or moremessenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. Without being bound by theory, miRNAs arefirst transcribed as primary transcripts or pri-miRNA with a cap andpoly-A tail and processed to short, 70-nucleotide stem-loop structuresknown as pre-miRNA in the cell nucleus. This processing is performed inanimals by a protein complex known as the Microprocessor complex,consisting of the nuclease Drosha and the double-stranded RNA bindingprotein Pasha. These pre-miRNAs are then processed to mature miRNAs inthe cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC).When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end. The remaining strand, knownas the anti-guide or passenger strand, is degraded as a RISC complexsubstrate. After integration into the active RISC complex, miRNAs basepair with their complementary mRNA molecules and induce mRNA degradationby argonaute proteins, the catalytically active members of the RISCcomplex. Animal miRNAs are usually complementary to a site in the 3′ UTRwhereas plant miRNAs are usually complementary to coding regions ofmRNAs.

The term ‘short hairpin RNA’ or ‘shRNA’ refers to RNA molecules havingan RNA sequence that makes a tight hairpin turn that can be used tosilence gene expression via RNA interference. The shRNA hairpinstructure is cleaved by the cellular machinery into siRNA, which is thenbound to the RNA-induced silencing complex (RISC). This complex binds toand cleaves mRNAs which match the siRNA that is bound to it. shRNA istranscribed by RNA Polymerase III whereas miRNA is transcribed by RNAPolymerase II. Techniques for designing target specific shRNA moleculesare known in the art.

An ‘expression vector’ is typically a nucleic acid molecule which is maybe integrating or autonomous, (i.e. self-replicating), and whichcontains the necessary components to achieve transcription of anexpressible sequence in a target cell, when introduced into the targetcell. Expression vectors may include plasmids, cosmids, phage, YAC, BAC,mini-chromosomes, viruses, e.g. retroviruses, adenovirus, lentivirus,SV-40, and the like; etc. Many such vectors have been described in theart and are suitable for use with the promoters of the presentinvention. Expression vectors of the present invention include apromoter as described herein, operably linked to an expressiblesequence, which may also be optionally operably linked to atranscription termination sequence, such as a polyadenylation sequence.The expression vector optionally contains nucleic acid elements whichconfer host selectivity, elements that facilitate replication of thevector, elements that facilitate integration of the vector into thegenome of the target cell, elements which confer properties, for exampleantibiotic resistance, to the target cell which allow selection orscreening of transformed cells and the like. Techniques and methods fordesign and construction of expression vectors are well known in the art.

It may be desirable, when driving expression of an expressible sequencewith a particular promoter system, to have the expression occur in astable and consistent manner. A factor that has been shown to affectexpression is the site of integration of an expression vector orconstruct into the genome of the target cell, sometimes called ‘positioneffects’. Such position effects may be caused by, for example, localchromatin structure which affects expression of sequences from thatregion of the genome. One method to control for position effects whenintegrating an expression vector or construct into the genome of atarget cell is to include a ‘genomic targeting sequence’ in the vectoror construct that directs integration of the vector or construct to aspecific genomic site. As an example, the hypoxanthinephosphoribosyltransferase (HPRT) gene has been used successfully forthis purpose (Bronson et al. 1996; Jasin et al. 1996). The HPRT gene hasadditional advantages as a genomic targeting sequence, for instance itsconcomitant use as a selectable marker system. Other genomic targetingsequences that may be useful in the present invention are described inthe art, for instance (Jasin et al. 1996; van der Weyden et al. 2002).The genomic targeting signals as described herein are useful in certainembodiments of the present invention.

Introduction of nucleic acids or expression vectors may be accomplishedusing techniques well known in the art, for example microinjection,electroporation, particle bombardment, or chemical transformation, suchas calcium-mediated transformation, as described for example in Maniatiset al. 1982, Molecular Cloning, A laboratory Manual, Cold Spring HarborLaboratory or in Ausubel et al. 1994, Current protocols in molecularbiology, Jolm Wiley and Sons.

S100B Promoters

The present invention herein provides novel S100B mini-promotersequences which are capable of effecting transcriptional expression in aspatial and temporal fashion similar to naturally occurring S100Bpromoters. The S100B mini-promoters of the invention comprise S100Bpromoter elements joined in a non-native configuration, thus providingadvantageous characteristics. Also provided are novel expression vectorcompositions comprising S100B mini-promoters which allow consistentspecific spatiotemporal transcription of expression sequences. Alsoprovided are novel methods utilizing these S100B mini-promoters andexpression vectors.

The S100B promoters of the invention, as described herein, are referredto as ‘mini-promoters’ to reflect the fact that the mini-promoterscomprise S100B promoter elements that are joined in a non-nativeconfiguration. In this context, native intervening sequences betweenpromoter elements may have been partially or completely removed, andoptionally may have been replaced with non-native sequences. In such afashion, the natural spacing of the promoter elements, for instance thehuman S100B regulatory element corresponding to SEQ ID NO: 2 and thehuman S100B basal promoter element corresponding to SEQ ID NO: 3, orsequences with substantial functional and/or sequence equivalence, isaltered. An advantage of such non-native mini-promoters is that theremoval of native intervening sequences reduces the size of themini-promoter while maintaining the functional activity of the promoter,thus improving the utility of the mini-promoter for variousapplications.

The inventors have demonstrated, as illustrated in the non-limitingWorking Examples, that a human S100B mini-promoter having a sequencecorresponding to SEQ ID NO: 1, and which is comprised of a human S100Bregulatory element having a nucleic acid sequence corresponding to SEQID NO: 2 operably linked in a non-native conformation to a human S100Bbasal promoter having a nucleic acid sequence corresponding to SEQ IDNO: 3, is capable of directing expression of an expressible sequencewhich is operably linked downstream of the S100B promoter in specificcell types in different regions of the brain. The S100B regulatoryelement (SEQ ID NO: 2) and S100B basal promoter element (SEQ ID NO: 3)have sequences which are identical to those found upstream of the humanS100B gene, found on chromosome 21 of the human genome. To place thesesequences in context, SEQ ID NO: 2 corresponds to absolute genomiccoordinates chr21:46864758-46867265 (strand-), while SEQ ID NO: 3corresponds to absolute genomic coordinates chr21:46849383-46850766(strand-), where the genomic coordinates are derived from NCBI Build36.1 human genome assembly of March 2006. In relative terms, SEQ ID NO:2 corresponds to position −17882 to −15294, while SEQ ID NO: 3corresponds to position −1302 to +81, where the position correspondingto +1 is defined as the first nucleic acid of exon 1 of the human S100Bgene. It is within the skill of one in the art to locate and determinethese relative positions based on published sequence information forthis gene, for instance found in the GenBank or PubMed public databases.It is understood that these genomic coordinates and relative positionsare provided for the purposes of context, and that if any discrepanciesexist between published sequences and the sequence listings providedherein, then the sequence listings shall prevail.

Promoters of the present invention may be modified with respect to thenative regulatory and/or native basal promoter sequence. In general,such modifications will not change the functional activity of thepromoter with respect to cell-type selectivity; and to the rate oftranscription in cells where the promoter is active. The modifiedpromoter provide for a transcription rate in a cell of interest of anexpressible sequence operably linked to a modified promoter sequencethat is at least about 75% the transcription rate of the promotersequence of SEQ ID NO:1, at least about 80%, at least about 90%, atleast about 95%, at least about 99%, or more. Methods of assessingpromoter strength and selectivity are known in the art, including, forexample, expression of a reporter sequence in a cell in vivo or invitro, and quantitating the reporter activity.

Modifications of interest include deletion of terminal or internalregions, and substitution or insertion of residues. Applicants haveidentified 38 conserved sequences in the S100B promoter (Table 1), where27 such conserved sequences are present in the regulatory element, and11 conserved sequences are present in the basal promoter. A promoter ofinterest in the present invention comprises at least 30, usually atleast 35, and may comprise all 38 of the identified conserved sequences.Sequences set forth in SEQ ID NO:1 that are not conserved may be deletedor substituted, usually modifications that retain the spacing betweenconserved sequences is preferred. In general the spacing between theregulatory element and the basal promoter is not more than about 10 KB,generally not more than about 1 KB, usually not more than about 500 nt,and may be not more than about 100 nt, down to a direct joining of thetwo sequences.

TABLE 3 List of conserved sequences in SEQ ID NO: 1. Start (relative toEnd (relative to SEQ ID NO: 1) SEQ ID NO: 1) Invariant sequence type 85105 Conserved sequence 141 160 Conserved sequence 212 231 Conservedsequence 317 344 Conserved sequence 415 447 Conserved sequence 495 514Conserved sequence 531 553 Conserved sequence 562 582 Conserved sequence603 624 Conserved sequence 651 710 Conserved sequence 720 746 Conservedsequence 806 829 Conserved sequence 931 966 Conserved sequence 1191 1210Conserved sequence 1245 1287 Conserved sequence 1300 1341 Conservedsequence 1350 1375 Conserved sequence 1422 1445 Conserved sequence 15481577 Conserved sequence 1630 1658 Conserved sequence 1708 1730 Conservedsequence 1743 1783 Conserved sequence 1786 1807 Conserved sequence 19121938 Conserved sequence 2088 2158 Conserved sequence 2212 2231 Conservedsequence 2315 2341 Conserved sequence 2586 2624 p53 validated bindingsite 2753 2792 Conserved sequence 2931 3010 Conserved sequence 3069 3092Conserved sequence 3276 3299 Conserved sequence 3332 3351 Conservedsequence 3387 3440 Conserved sequence 3456 3475 Conserved sequence 34823653 Conserved sequence 3661 3680 Conserved sequence 3699 3725 Conservedsequence The start and end coordinates of the sequences are relative tothe full SEQ ID NO: 1 sequence.

In some embodiments of the invention, there is thus provided an isolatednucleic acid fragment comprising an S100B mini-promoter, wherein theS100B promoter comprises an S100B regulatory element operably linked ina non-native conformation to an S100B basal promoter. In certainembodiments of the invention, the S100B promoter may have a nucleic acidsequence which is substantially similar in sequence and function to SEQID NO: 1. In some embodiments, the S100B regulatory element may have anucleic acid sequence which is substantially similar in sequence andfunction to SEQ ID NO: 2. In some embodiments, the S100B basal promotermay have a nucleic acid sequence which is substantially similar insequence and function to SEQ ID NO: 3. The S100B promoter may further beoperably linked to an expressible sequence, e.g. reporter genes, genesencoding a polypeptide of interest, regulatory RNA sequences such asmiRNA, siRNA, anti-sense RNA, etc., and the like. Reporter genesequences include, for example luciferase, beta-galactosidase, greenfluorescent protein, enhanced green fluorescent protein, and the like asknown in the art. The expressible sequence may encode a protein ofinterest, for example a therapeutic protein, receptor, antibody, growthfactor, and the like.

It is an object of the present invention to provide means of expressinga gene, protein, RNA interference molecule or the like in a cell, tissueor organ. As such, the inventors thus provide novel expression vectorscomprising S100B mini-promoters which are capable of accomplishing thistask. In some embodiments of the invention, there is provided aexpression vector comprising an S100B promoter element, wherein theS100B promoter element comprises an S100B regulatory element operablylinked in a non-native conformation to an S100B basal promoter element.The S100B promoter element may have a nucleic acid sequencesubstantially similar in sequence and function to SEQ ID NO: 1. TheS100B regulatory element may have a nucleic acid sequence substantiallysimilar in sequence and function to SEQ ID NO: 2. The S100B basalpromoter element may have a nucleic acid sequence substantially similarin sequence and function to SEQ ID NO: 3. The S100B promoter may furtherbe operably linked to an expressible sequence, e.g. reporter genes,genes encoding a polypeptide of interest, regulatory RNA sequences suchas miRNA, siRNA, anti-sense RNA, etc., and the like. Reporter genesequences include, for example luciferase, beta-galactosidase, greenfluorescent protein, enhanced green fluorescent protein, and the like asknown in the art. The expressible sequence may encode a protein ofinterest, for example a therapeutic protein, receptor, antibody, growthfactor, and the like. The expression vector may further comprise agenomic targeting sequence. The genomic targeting sequence may be HPRT.

The inventors have herein demonstrated that expression vectorscomprising novel S100B mini-promoter elements are capable of directingtranscription of an expression sequence in specific cell types, mostnotably glial cells, in specific regions of the brain. In someembodiments of the invention, there is thus provided a method forexpressing a gene, protein, RNA interference molecule or the like in thetargeted cells of the brain. Cells of interest include, withoutlimitation, cells of the peripheral or central nervous system andprogenitors thereof, e.g. embryonic stem cells, neural stem cells,neurons, glial cells, astrocytes, microgial cells, etc. The methodcomprises introducing into a glial cell or progenitor cell thereof anexpression vector comprising an S100B mini-promoter element, wherein theS100B mini-promoter element comprises an S100B regulatory elementoperably linked in a non-native conformation to an S100B basal promoterelement. The S100B promoter element may have a nucleic acid sequencesubstantially similar in sequence and function to SEQ ID NO: 1. TheS100B regulatory element may have a nucleic acid sequence substantiallysimilar in sequence and function to SEQ ID NO: 2. The S100B basalpromoter element may have a nucleic acid sequence substantially similarin sequence and function to SEQ ID NO: 3. The S100B promoter may furtherbe operably linked to an expressible sequence, e.g. reporter genes,genes encoding a polypeptide of interest, regulatory RNA sequences suchas miRNA, siRNA, anti-sense RNA, etc., and the like. Reporter genesequences include, for example luciferase, beta-galactosidase, greenfluorescent protein, enhanced green fluorescent protein, and the like asknown in the art. The expressible sequence may encode a protein ofinterest, for example a therapeutic protein, receptor, antibody, growthfactor, and the like. The expression vector may thus further comprise agenomic targeting sequence. The genomic targeting sequence may be HPRT.

In other embodiments of the invention, there is provided a method foridentifying or labeling a cell, the method comprising introducing intothe cell a expression vector comprising an S100B mini-promoter elementoperably linked to an expressible sequence, wherein the S100Bmini-promoter element comprises an S100B regulatory element operablylinked in a non-native conformation to an S100B basal promoter element,and wherein the expressible sequence comprises a reporter gene. TheS100B promoter element may have a nucleic acid sequence substantiallysimilar in sequence and function to SEQ ID NO: 1. The S100B regulatoryelement may have a nucleic acid sequence substantially similar insequence and function to SEQ ID NO: 2. The S100B basal promoter elementmay have a nucleic acid sequence substantially similar in sequence andfunction to SEQ ID NO: 3. The inventors have demonstrated thatexpression vectors comprising certain human S100B promoter elements arecapable of expression in glial cell types in specific regions of thebrain. In some embodiments, the cell is a peripheral or central nervoussystem cell or progenitors thereof, including, without limitation,embryonic stem cells, neural stem cells, glial cell, e.g. Bergmann glialcells, astrocytes, and the like. Reporter gene sequences include, forexample luciferase, beta-galactosidase, green fluorescent protein,enhanced green fluorescent protein, and the like as known in the art.The expressible sequence may encode a protein of interest, for example atherapeutic protein, receptor, antibody, growth factor, RNA interferencemolecule and the like.

In further embodiments of the invention, there is provided a method formonitoring or tracking the development or maturation of a cell in theglial cell lineage. The method comprises: 1) introducing into aprogenitor to a glial cell, e.g. an embryonic stem cells, neural stemcell, glial progenitor cell, glial cell, etc., cell an expression vectorcomprising an S100B mini-promoter element operably linked to anexpressible sequence, wherein the S100B mini-promoter element comprisesan S100B regulatory element operably linked in a non-native conformationto an S100B basal promoter element, and wherein the expressible sequencecomprises a reporter gene; and 2) detecting the expression of thereporter gene glial cell progeny of the cells as a means of determiningthe lineage, identity or developmental state of the cell or cellprogeny. In such a fashion, one may be able to follow the development ofa parent cell as it differentiates into more mature cells. As anexample, one could introduce a expression vector comprising theaforementioned S100B promoter elements into a pluripotent stem cell,monitor the expression of the reporter gene that is being expressed bythe S100B promoter elements during the maturation and differentiation ofthe stem cell and thus determine the state of maturation, for instancein the differentiation of the pluripotent stem cell into a glial cell.The inventors have demonstrated that the S100B promoter elementsdescribed herein cause transcriptional expression in certain glial celltypes, and so detection of reporter gene expression in a cell would thusbe indicative of the cellular identity of the cell as being a glialcell.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Working Examples General Methods

Expression vector. The nucleic acid fragment corresponding to SEQ ID NO:1 was inserted into the multiple cloning site of the pEMS1306 (seeFIG. 1) to produce the expression vector S100B-C.

Derivation of mEMS1204 embryonic stem cells. Blastocysts were obtainedfrom natural mating of B6-Hprt1^(b-m3) females to 129-ROSA26 males at3.5 dpc. Blastocysts were flushed from uterine horns as per (Hogan etal. 1994), cultured in EmbryoMax® KSOM with 1/2 Amino Acids, Glucose andPhenol Red (Cat #MR-121, Millipore/Chermicon, Temecula, Calif.) for 3-5h, and then transferred onto mitomycin C (mitC; Cat#M4287, Sigma,Oakville, ON) mitotically inactivated B6-Hprt1^(b-m3), B6129F1, or 129mouse embryonic feeders (MEFs) derived from 13.5-day post-coital embryos(Ponchio et al. 2000) in 96-well plates containing KSR-ESC (Knockout™D-MEM, Cat#10829-018, Invitrogen, Burlington, ON) with 2 mM L-glutamine(Cat#25030-081, Invitrogen, Burlington, ON), 0.1 mM MEM nonessentialamino acid solution (Cat#11140-050, Invitrogen, Burlington, ON) and 16%Knockout™ Serum Replacement (Cat#10828-028, Invitrogen, Burlington, ON))media (MEF media was replaced 3-5 hour prior to transfer).

Blastocysts were cultured as per (Cheng et al. 2004) with the followingmodifications: Cells were cultured for 7-9 days in KSR-ESC with minimaldisturbance (checked on day 2 to determine if the blastocysts had‘hatched’ out of the zona pellucida) and no media changes. Blastocystswhich hatched and had a well developed ICM (inner cell mass) weretreated with 20 μl 0.25% trypsin-EDTA (Invitrogen, Burlington, ON) for 5min at 37° C., triturated with a 200 μl pipetman, inactivated with 30 μl0.5 mg/ml soybean trypsin inhibitor (Invitrogen, Burlington, ON), andbrought up to 200 μl with KSR-ESC, then transferred individually to a24-well MEF plate containing 1800 μl KSR-ESC, for a total volume of 2ml:

Beginning 4 days later, KSR-ESC media was replaced with FBS-ESC media(DMEM (Cat #11960-069, Invitrogen, Burlington, ON) with 2 mM L-glutamine(Invitrogen, Burlington, ON), 0.1 mM MEM nonessential amino acidsolution (Invitrogen, Burlington, ON), 16% ES Cell Qualified fetalbovine serum (FBS, Invitrogen, Burlington, ON) and 0.01%β-mercaptoethanol (Sigma, Oakville, ON) in 25%, 50%, 75% proportions(respectively) to adapt the cells to FBS containing media.

On day 7 the cells were trypsinized to one well of a 24 well platecontaining 1 ml of 100% FBS-ESC media, with daily media replacement.Once confluent, wells containing ESC colonies were expanded 3×24 wells(with MEFs), then passaged to 3×24 (with MEFs) and 3×12 well (plastic—noMEFs) for DNA analysis. Once confluent, the 3×24 wells were combined,aliquoted (3 vials), and frozen in ESC-freeze media (50% FBS, 40%FBS-ESC media, 10% DMSO (Sigma, Oakville, ON), and the 3×12 well treatedwith lysis buffer (Fisher Scientific, Ottawa, ON), mixed and aliquoted.Cultures were genotyped for X & Y chromosomes (Clapcote and Roder 2005),Gt(ROSA)26Sor^(tm1Sor) and WT alleles and Hprt1^(b-m3) and WT alleles.B6129F1-Gt(ROSA)26Sor^(tm1Sor)/+, Hprt1^(b-m3)/Y andB6129F1-Gt(ROSA)26Sor^(tm1Sor)+l+, Hprt1^(b-m3)/Y cell lines wereidentified.

Knock-in at the Hprt1 locus. The S100B-C plasmid DNA was purified withQiagen Maxi Kit (Qiagen, Mississauga, ON), resuspended in 10:1 Tris-EDTA(TE, pH7.0) buffer, and linearized with I-SceI (New England Biolabs,Pickering, ON). Linearized plasmid DNA was resuspended in 85 μl of TE(10:0.1) to a final concentration of 187.5 ng/μl. mEMS1204 ESCs weregrown to confluence on 4-6 T75 flasks of mitC treated Hprt1^(b-m3) mouseembryonic feeders (MEFs) in FBS-ESC media. ESCs (1.7−2.5×10⁷) in 720 μl1×PBS were added to the linearized DNA and electroporated in a 4 mmelectroporation cuvette (Bio-Rad Genepulser, Mississauga, ON), at 240 V,50 μF, 6-10 msec pulse, immediately resuspended in a total volume of 5ml of FBS-ESC media and plated onto 5×100 mm dishes of mitC B6129F1 MEFsin a total volume of 12 ml/100 mm dish. 24-36 h post-electroporation,correctly targeted homologous recombinants were selected for using HATmedia (FBS-ESC media containing 1×HAT ((0.1 mM sodium hypoxanthine, 0.4mM aminopterin, 0.16 mM thymidine), Cat#21060-017, Invitrogen,Burlington, ON). HAT media was changed every day for the first 3 days,and then every 3^(rd) day thereafter, for up to 10 days. Individualcolonies were counted and, typically, no more than 2 isolated colonieswere picked per 100 mm dish to optimize for independent homologousrecombination events. These colonies were expanded under standardprotocols for verification of the desired recombination event.

Derivation of knock-in mice. Chimeric mice from untargeted and targetedESCs were generated by microinjection (Hogan et al. 1994) into B6(E14TG2a derived) and B6-Alb (E14TG2a and mEMS1204 derived) E3.5blastocysts, or co-culture (Lee et al. 2007) with diploid ICR (CharlesRiver, Wilmington Wash. Stock#022) E2.5 morula (cultured overnight tothe blastocyst stage), followed by implantation into the uterine hornsof 2.5 day pseudopregnant ICR females. Chimeras were identified and coatcolor chimerism determined as outlined below.

Male chimeras derived from the E14TG2a cell lines were mated with B6 orB6-Alb females, and germline transmission was identified in the formercase by the transmission of the dominant A^(w) (nonagouti; white belliedagouti) allele, making the progeny appear brown with a cream belly, orin the later case by the combination of A^(w) and Tyr^(c-ch)(tyrosinase; chinchilla), making the progeny appear golden. Non-germlineprogeny from the cross to B6 were homozygous for the recessive a(nonagouti; nonagouti) allele and appeared black, whereas non-germlineprogeny from the cross to B6-Alb were homozygous for the recessiveTyr^(c) (tyrosinase; albino) allele and appeared white.

Male chimeras derived from the mEMS1204 cell lines were mated withB6-Alb females, and germline transmission identified by the presence ofthe dominant Tyr⁺ (tyrosinase; wild type) and the A^(w) (nonagouti;white bellied agouti) or a (nonagouti; nonagouti) alleles making theprogeny appear brown with a cream belly or black, respectively.Non-germline progeny were homozygous for the recessive Tyr^(c-2J)(tyrosinase; albino 2 Jackson) allele and appear white. All germlinefemale offspring should carry the knock-in X Chromosome and were matedwith B6 males. N2 offspring were analyzed for the presence of the KIallele by PCR.

Determination of coat color chimerism. E14TG2a- and mEMS1204-derivedchimeras were identified and level of coat color chimerism determined asfollows. E14TG2a ESCs, homozygous for A^(w) and Tyc^(c-ch) as they arederived from the 129/OlaHsd strain (Hooper et al. 1987a; Hooper et al.1987b), will produce chimeras with cream/chinchilla and agouti patcheson a black background when injected into B6 blastocysts. Thecream/chinchilla patches result from melanocytes derived solely from theESCs (A^(w)/A^(w), Tyr^(c-ch)/Tyr^(c-ch)), whereas agouti patches resultfrom melanocytes that are a mixture of ESC (A^(w)/A^(w),Tyr^(c-ch)/Tyr^(c-ch)) and host (a/a, Tyr⁺/Tyr⁺). However, E14TG2a ESCs,when injected into B6-Alb (a/a, Tyr^(c)/Tyr^(c)) produce chimeras withchinchilla and light chinchilla coat color patches on a whitebackground. The former is derived solely from the ESCs (A^(w)/A^(w),Tyr^(c-ch)/Tyr^(c-ch)), whereas the latter is again a mix of the ESC(A^(w)/A^(w), Tyr^(c-ch)/Tyr^(c-ch)) and host (a/a, Tyr^(c)/Tyr^(c)).mEMS1204-derived chimeras were identified and coat color chimerismdetermined in the same manner.

mEMS1204 ESCs, heterozygous A^(w)/a and homozygous for the wild typeTyr⁺ alleles will produce chimeras with agouti and black patches on awhite background when injected into B6-Alb blastocysts. The agoutipatches result from melanocytes derived solely from the ESCs (A^(w)/a,Tyr⁺/Tyr⁺), whereas ‘black’ patches result from melanocytes that are amixture of ESC (A^(w)/a, Tyr⁺/Tyr⁺) and host (a/a,Tyr^(c-2J)/Tyr^(c-2J)).

For E14TG2a injections into B6 and mEMS1204 injections into B6-Alb,overall chimerism was calculated by summing the percent of coat colorpatches derived solely from the ESC, plus half the percent of the ESC+host areas, where we conservatively estimated that half the melanocytesderive from the ESC and half from the host. For E14TG2a injections intoB6-Alb, the similarity between chinchilla and light chinchilla on awhite background presented difficulty when attempting to estimateoverall coat color chimerism. As such, we estimated the percentchimerism based solely on the total chimerism observed when compared toa white mouse, resulting in slightly inflated overall percent chimerismfor this small cohort of mice.

Immunohistochemistry and Immunofluorescence. Adult male chimeric and agematched control mice were perfused with 4% paraformaldehyde (PFA) aspreviously described (Young et al. 2002). Whole brains were dissectedout and post-perfusion immersion fixed with PFA for 2-3 hours at 4° C.Brains were then transferred to 20% sucrose at 4° C. overnight withgentle shaking. The brains were cryostat sectioned sagittally at 12-14μm and mounted on superfost-plus slides (Cat#12-550-15, ThermoFisherScientific, Waltham, Mass.). EGFP expression was detected by directfluorescence of EGFP or by indirect immunofluorescence with anti-GFPantibodies (Abcam, Cambridge, Mass.) using a BioRad confocal laserscanning microscope (CLSM, BioRad, Hercules, Calif.).

For double label immunofluorescence analyses to determine cell types inthe cerebellum, anti-GFAP was used in conjunction with direct EGFPfluorescence and imaged by CLSM (Liu et al. 2007). In brief, slidemounted brain sections, were permeabilized with phosphate bufferedsaline containing 0.1% triton-X100 (PBST), blocked with PBST containing5% normal horse serum and 1% BSA, then incubated with primary antibodiesovernight at room temperature in a humid chamber. Following three washeswith PBST, the tissue were incubated with secondary antibodies (goatanti-rabbit-Alexa-594 conjugate, Molecular Probes, Eugene, Oreg.). Theslides were counterstained with TOTO3/DAPI (1 μM each) for labeling allnuclei in confocal images. Bright field analyses were also conductedfollowing immunocytochemical detection of anti-GFP using the VectastainABC kit and DAB as the chromogen to give a brown reaction productfollowing the manufacturer's directions. Bright field images werevisualized on a Zeiss Axiovert microscope and Axiovision Software (CarlZeiss Microimaging, Thornwood, N.Y.).

Selection of S100B promoter elements. Cross-species comparisons, orphylogenetic footprinting, were identified as a means to predictregulatory regions. The two mammalian species with the best evolutionarydistance to use this approach are human and mouse. In the specific caseof S100B, we computed the conservation level between human and mouse,taking into consideration the non-coding sequence located between theend of the upstream gene (PRMT2) and the end of S100B, including allintron sequences. Due to a lot of repeats in this area, the conservationis sparse and we set up a threshold of 54% of identity to select ourcandidate regulatory regions (FIG. 1). The S100B basal promoter (SEQ IDNO: 3) and regulatory region (SEQ ID NO: 2) were chosen based on thesecriteria.

Expression of reporter in glial cells by S100B-C promoter element. TheS100B-C DNA expression vector (FIG. 2) comprising the S100B promoterelement corresponding to SEQ ID NO: 1 (which is itself comprised of SEQID NO: 2 linked to SEQ ID NO: 3) was introduced into mouse embryonicstem cells (ESCs) at the HPRT locus. The ESCs were used to generategenetically modified mice containing S100B-C. Immunohistochemical andimmunofluorescence analysis of mouse brain tissue slices revealed EGFPreporter expression in the Bergmann glial cells of the cerebellum (FIG.3).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

REFERENCES

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1. An isolated polynucleotide comprising an S100B regulatory elementoperably joined to an S100B basal promoter through a non-native spacingbetween the promoter and the regulatory element.
 2. The isolatedpolynucleotide of claim 2, operably linked to an expressible sequence.3. A vector comprising the isolated polynucleotide of claim 1 or claim2.
 4. A cell comprising the vector of claim
 3. 5. The cell of claim 4,wherein the vector is stably integrated into the genome of the cell. 6.The cell of claim 4 or claim 5, wherein the cell is a stem cell.
 7. Amethod of expressing a sequence of interest, the method comprisingoperably linking the sequence of interest to the polynucleotide of claim1; and introducing into a cell permissive for expression from the S100Bpromoter.